We broadly investigate the mechanisms used in cells to regulate gene expression at the translational level. Current research is focused on the question of how ribosomes are disassembled following the completion of translation at stop codons. Without this recycling process, ribosomes would quickly accumulate at stop codons, limiting the cell's ability to make new protein. The lab primarily employs high-throughput sequencing methods, such as mRNA-Seq and ribosome footprint profiling with computational analysis. We also use an array of biochemical approaches, such as western blots and reporter assays, to complement this work. Finally, we are developing tools for imaging single polysomes in living yeast and mammalian cells, using fluorescence microscopy and the suntag system. Recycling begins when the stop codon is decoded by the canonical release factors, eRF1 and eRF3. Following GTP hydrolysis by eRF3, the ATPase Rli1 (ABCE1 in higher eukaryotes) separates the two subunits from each other. In prior work, we established that lack of Rli1 in the cell leads to a surprising accumulation of ribosomes in the 3'UTR and the translation of short open reading frames. The recycling factor ABCE1 is known to be upregulated in many types of cancer, suggesting that ribosome recycling is critical in cancer cells, potentially as a way to ensure an adequate supply of recycled ribosomes for new rounds of translation during rapid proliferation. We believe this process to also be critical during the innate immune response because the activity of ABCE1 is thought to modulated by genes that are upregulated by interferon. A better understanding of the mechanism of ribosome recycling is therefore important for overcoming major challenges to human health. We have recently investigated the yeast factors Tma64, Tma20, and Tma22 (eIF2D, MCT-1, and DENR in mammals). Our work has now strongly suggested that these factors are required for ribosome recycling. Without them, we have found that ribosomes enter 3'UTRs and reinitiate new translation, likely by at least two mechanisms. First, we found evidence (ribosome profiling peaks on 3'UTR AUG codons and via western blot of reporter proteins) that that these factors promote recycling of 40S ribosomes. However, we also observed evidence of 80S reinitiation and stop codon readthrough. Our research has therefore shown that these factors play a critical role in preventing translation of 3'UTRs at multiple levels. Mutation of these factors has been shown to be associated with autism and cancer in humans, suggesting that the peptides produced in their absence in yeast may be linked to these diseases. We have begun work on deciphering the rules and factors involved in ribosome reinitiation downstream of the stop codon by using masspec and ribosome footprint profiling approaches. Our research on reporter constructs suggests that 80S ribosomes are readily capable of reinitiating translation following peptide release over a long region of the 3'UTR adjacent to the stop codon. Therefore, components of the recycling machinery such as Rli1, Tma64, Tma20, and Tma22 are critical to prevent this aberrant process from occurring. We are also exploring potential biological roles for 3'UTR ribosomes and the possibility that loss of efficient recycling under some cellular environments can alter fitness. We are examining the effects of nutrient deprivation stress (yeast) and simulated viral infection (human cell lines), for example, in reducing the efficiency of termination and recycling. Our results now suggest many stresses induce translation of 3'UTRs but that the mechanism involved is variable (40S reinitiation, 80S reinitiation, readthrough, etc.), implying that the repertoire of 3'UTR peptides that is translated depends on the stress. We have also shown that we can stimulate the cell's antiviral response and that levels of 3'UTR ribosomes increase when this occurs. This finding implies that factors activated during the innate immune response directly affect ribosome recycling and that peptides produced may be immunogenic.